Our goal is to define the enzymatic properties of the Ras Converting Enzyme (Rcelp), a protease that is required for CaaX protein biosynthesis. CaaX proteins are lipidated molecules that often function as signaling molecules in important cellularpathways. Ras and RhoB are key examples. Because of the role that CaaX proteins have in cellular transformation (e.g., activated forms of Ras are associated with 30% of all cancers), strategies that regulate the biosynthesisof these proteins are being explored as novel anti- cancer therapies. Rcelp is a new target in these strategies. Rcelp is an atypical protease, having multiple membrane spans and lacking a canonical protease motif. To date, the mechanism of Rcelp remains undefined. We hypothesize that the Rcelp active site is comprised of a subset of residues that are invariably conserved between Rcelp orthologs. In our preliminary studies, which take advantage of the S. cerevisiae system, we have 1) identified four residues that are critically important for Rcelp function, 2) discovered a novel compound that is potentially a specific and irreversible inhibitor of Rcelp, and 3) utilized a dual genetic/ biochemical reporter and deletion analysis to assess the topology and importance of the Rcelp C-terminus. We have developed a coherent set of biochemical, genetic, chemical, and molecular approaches around our findings that will be used for defining the active site and enzymatic properties of Rcelp. Specifically, we will use a novel quantitative genetic assay to detail the importance of charge and position for residues deemed critical for enzymatic activity and to evaluate the substrate specificity of Rcelp mutants. We will use an in vitro assay that monitors cleavage of a quenched fluorescent peptide substrate to evaluate the effect of novel inhibitors and mutations on the kinetic parameters of Reel p.mutants. Finally, we will use a dual genetic/biochemical topology reporter and deletion approaches to identify the functional domains of Rcelp. In sum, this proposal will clarify the enzymatic properties of Rcelp, a member of an emerging class of multi-span membrane-boundproteases having biomedical importance.